Anal. Chem. 1980, 52, 1239-1245
T h e R2PI method should be valuable for detection of these species. T h e studies described in this report were not designed to establish the ultimate detection limits obtainable by RZPI in conjunction with gas chromatography. Nonetheless, detection of PAH compounds a t levels down to 10 pg was easily achieved. Improvement by a factor of ten can be expected given state-of-the-art chromatographic technique and an additional factor of 100-1000 may be realized given an improved laser system. ACKNOWLEDGMENT The authors express their apprecitation to Jerry Gelbwachs a n d Richard Keller for helpful suggestions concerning the manuscript. LITERATURE CITED Frueholz, R.; Wessel, J.; Wheatley, E. Anal. Chem. 1980, 52, 281-284. Brophy, J. H.; Rettner. C. T. Opt. Lett. 1979, 4 , 337-339. Brophy, J. H.; Rettner, C . T. Chem. Phys. Lett. 1979, 67,351-355. Antonov, V. S.;Knyazev, I . N.; Letokhov, V. S.;Matiuk, V. M.; Movshev, V. G.; Potapov, V. K. Opt. Lett. 1978, 3 , 37-39. Held, B.; Mainfray, G.; Manus, C.; Morellec, J.; Phys. Rev. Lett. 1972, 28, 130-131. Lineberger. W. C.; Patterson, T. A. Chem. Phys. Lett. 1972, 13. 40-44. Johnson, P. M.; Berman, M. R.; Zakheim, D. J. J . Chem. Phys. 1975, 62, 2800-2502. Johnson, P. M. J . Chem. Phys. 1975, 62, 4562-4563. Johnson, P. M. J . Chem. Phys. 1976, 64, 4638-4644. Johnson, P. M. Acc. Chem. Res. 1980, 73, 20-26. Petty. Gena; Tai, C.: Dalbv. F. W. Phys. Rev. Lett. 1975, 34, 1207-1209. Parker, D. H.; Sheng, S. J.; El-Sayed, M. A. J . Chem. Phys. 1976, 65, 5534-5535. El-Sayed, M. A. Chem. Phys. Lett. 1978, 56, Parker, D. H.; Berg, J. 0.; 197- 199.
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(14) Berg, J. 0.; Parker, D. H.; El-Sayed, M. A. Chem. Phys. Lett. 1978, 56, 411-413. (15) Parker, D. H.; El-Sayed, M. A. Chem. Phys. 1979, 42, 379-387. (16) Parker, D. H.; Pandolfi, R.; Stannard, P. R.; El-Say&, M. A. Chem. Phys. 1980, 45, 27-37. (17) Feldman, D. L.; Lengl, R. K.;Zare, R. N. Chem. Phys. Lett. 1977, 52, 4 13-41 7. (18) Lubman, D. M.; Naaman, R.; Zare, H. N. J . Chern. Phys., in press. (19) Herrmann, A.; Leutwyler, S.; Schumacher, E.; Woste, L. Chem. Phys. Lett. 1977, 52, 418-425. (20) Zandee, L.; Bernstein, R. B.; Lichtin, t). A. J . Chem. Phys. 1978, 69, 3427-3429. (21) Zandee, L.; Bernstein, R. B. J . Chem. Phys. 1979, 70, 2574-2575. (22) KrogMespersen, K.; Rava, R. P.; Goodman, L. Chem. Phys. Left. 1979, 64. 413-416. (23) KroghJespersen, K.; Rava, R . P.; Goodman, L. Chem. Phys. 1979, 44, 295-302. (24) Williamson, A. D.; Compton, R. N. Chem. Phys. Lett. 1979, 62, 295-297. (25) Williamson, A. D.;ComDton, R. N.; Eland, J. H. D. J . Chem. Phys. 1979. 70,590-591. (26) Hurst, G. S.; Nayfeh, M. H.; Young, J. P. Appl. Phys. Lett. 1977, 30, 229-231 ~ -. . (27) Hurst, G. S.; Payne, M. G.; Kramer, S.D.; Young, J. P. Rev. Mod. Phys. 1979. 51. 767-819. (28) Driscoll, J. N.; Ford, J.; Jaramlllo, L.; Becker. J. H.; Hewitt, G.; Marshall, J. K.; Onishuk, F. Am. Lab. 1978, 70(5),137-147. (29) Driscoll, J. N.; Ford, J.; Jaramillo, L. F.; Gruber, E. T. J . Chromatogr. 1978, 158, 171-180. (30) Hansch, T. W. Appl. Opt. 1972, 7 7 , 895.
RECEIVED for review January 21,1980. Accepted April 8,1980. This work was supported in part by the U S . Air Force Office of Scientific Research under Grant AFOSR-77-3438, by the National Science Foundation under Grant CHE77-16074, and by t h e Department of Energy, Contract DE-AC0379EV10239.
Sample Preparation and Gas Chromatography-Mass Spectrometry Determination of 2,3,7,8-Tetrachlorodibenzo-p-dioxin R. L. Harless,’ E. 0. Oswald, and M. K. Wilkinson U S .Environmental Protection Agency, Health Effects Research Laboratory, ETD, ACB, MD-69, Research Triangle Park, North Carolina 2771 1
A. E. Dupuy, Jr., D. D. McDaniel, and Han Tai
U.S. Environmental Protection Agency, OPIIIOTS, SAD, Field Studies Branch, Toxicant Analysis Center, Bay St. Louis, Mississ$pi 39529
Analytical extraction, clean-up procedures, and capillary column gas chromatography-high resolution mass spectrometry methods are presented for determination of low concentrations (pg/g levels) of 2,3,7,8-tetrachlorodibenz~p-dioxin (TCDD). The application of these procedures and methods in the analysis of human milk, beef liver, fish, water, and sediment is described. The TCDD methodology provided a mean % recovery of 80% for 2.5 to 10 ng 37CI,-TCDD, internal standard, and a mean % accuracy of f23% for 1 to 1250 pg TCDD in quality assurance samples. The precision of the GC-HRMS technique was determined to be f20 % relatlve to 2 to 10 pg TCDD quantification standards during daily operatlons.
T h e highly toxic compound, 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD), may be formed as a by-product in manufacturing processes utilizing tetrachlorobenzene to produce trichlorophenol. Under high pressure, high temperature, and
very basic conditions 1,2,4,5-tetrachlorobenzene is hydrolyzed to the 2,4,5-trichlorophenate. T h e chlorinated phenol is produced upon acidification. Unfortunately, a condensation can take place in this reaction which results in the formation of TCDD. Herbicides containing esters of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) manufactured from trichlorophenol have been found to contain trace amounts of TCDD. Recent reports have indicated chlorinated dibenzo-p-dioxins including TCDD may be formed in combustion processes. TCDD has been recognized as an extremely toxic (LDS00.6 pglkg, female guinea pig, most sensitive species) ( I ) , carcinogenic ( 2 ) ,and teratogenic ( 3 ) compound. Because of its toxicity and occurrence as a trace contaminant in chemical products, it is necessary to analyze for TCDD a t the low parts per trillion (ppt) concentration range, which is below the usual limits of detection for pesticide residue analysis. Methods for determining ppt TCDD in sample extracts using high resolution mass spectrometry (HRMS) ( 4 , 5 ) ,packed column gas chromatography (GC) in conjunction with low and high resolution MS (6-8), high resolution gas
This article not subject to U S . Copyright. Published 1980 by the American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 52, NO. 8, JULY 1980
chromatography with low resolution MS (9),and GC-negative ion chemical ionization MS (10) have been reported. T h e analysis of human, biological, and environmental samples for TCDD contamination in the p p t concentration range is complicated by the presence of many interfering components ranging from naturally occurring compounds t o industrial pollutants. Therefore, an extremely efficient and specific analytical clean-up procedure is a prerequisite for ppt T C D D analysis utilizing GC-MS detection techniques. T h e GC-MS detection technique must be ultra-sensitive and highly specific because of the required low ppt detection limits. High resolution glass capillary column GC interfaced with medium and high resolution MS specific mass detection provides the required GC resolution of components, MS sensitivity, and specificity for TCDD analysis in the 0.03 t o 100 ppt concentration range. This paper will describe the current sample preparation procedures and capillary column GC/HRMS techniques developed and applied by EPA laboratories for the isolation and quantitative determination of TCDD residues in human, biological, and environmental samples. TCDD results generated in the EPA laboratory and collaborating laboratories, are evaluated and released by designated EPA officials. Therefore, only the results of quality assurance samples and samples with unusual contamination are discussed in this paper.
EXPERIMENTAL Safety. TCDD is toxic and can pose grave health hazards if handled improperly. Techniques used for handling radioactive and infectious materials are applicable to TCDD. Only qualified individuals who are trained in laboratory procedures and are familiar with the hazards of TCDD should handle this substance. Females of childbearing age should not work with this material. A good laboratory practice involves routine physical examinations and blood checks of employees working with TCDD. Also facial photographs using oblique photoflood lighting should be periodically taken to detect chloroacne, which is an early indication of TCDD exposure. Instrumentation. A Varian Model 2700 GC interfaced with a Varian 311A MS was utilized for these analyses. The interface design (11) ensured maximum transfer efficiency. The GC was equipped with a 30 m X 0.25 mm i.d. SE-30 WCOT glass capillary column. The MS was equipped with a turbo-molecular vacuum pumping system, combination chemical ionization (CI) electron impact (EI) ion source (operated in the E1 mode), and a Varian eight-channel hardware (manual control) Multiple Ion Selection (MIS) device. The vacuum system easily accommodated the 5 mL/min helium flow from the GC/MS interface and did not contribute detectable background Contamination. The MIS device was operated in the normal coupled electric mode (jumping the acceleration voltage). Each MIS channel was equipped with individual controls for selecting a specific acceleration voltage, measuring range, out-put signal bandwidth, compensation for background contamination, and integration rate. The intensities of the selected masses were monitored in a time-division multiplex system, setting alternatively to each of the selected masses and recording their intensities simultaneously on an eight-channel Soltec recorder. The adjustable integration rate, 0.01 t o 1 s was sufficient to accurately reproduce capillary column peaks 2 s wide a t half height. Capillary Column GC/HRMS Multiple Ion Selection Analysis. The magnet current was tuned to perfluorokerosene (PFK; m / z 318.9793 and the MS was adjusted for 5000 to 9000 mass resolution. The ESA voltage was monitored and used in calculating the exact acceleration voltage required for the masses, m / z 327.8847 C12H40237C14, m / z 321.8936 C12H,0235C1$'C1,and m / z 319.8965 C12H40235C14 The calculated values were introduced to MIS channels 2, 3, and 4. Two microliters of quantification standard, 500 pg/pL 37C1-TCDD(labeled 2,3,7,8-tetrachlorodibenzo-p-dioxin, 37C1-TCDDisotopic purity greater than 98%) and 1 pg/pL TCDD, and 0.5 WLof n-tetradecane (keeper, improves chromatographic resolution) were injected into the capillary column (on column splitless injection). The GC parameters were:
column temperature 80 " C; 6 min isothermal then programmed at 34 OC/min to 265 "C; solvent vent closed at 14 min; MIS analysis initiated at 16 min; TCDD retention time 23 f 0.25 min. Other GC-HRMS parameters were: injection port 260 "C; GC transfer line 255 "C; ion source 240 "C; specific and variable acceleration voltage, 3 kV maximum; electron energy 70 eV; filament emission l mA; mass resolution 5000 to 10000; multiplier gain greater than lo6. COCl Loss Analysis. The magnet current was tuned to PFK m / z 254.9856 and the exact acceleration voltages required for TCDD masses m / z 256.9327, m / z 258.9298, m / z 319.8965, m / z 321.8936, and the m / z 327.8847 corresponding to 37C1-TCDD,were introduced into respective MIS channels. The analysis was performed adhering to the previously described time schedule of events. The GC-HRMS MIS five-channel simultaneous responses for 37C1-TCDDand TCDD were observed and recorded at the correct GC retention time for TCDD. Elemental Composition Analysis. The MS was adjusted for 10000 mass resolution utilizing PFK m / z 318.9793 as reference. The capillary column GC-HRMS peak matching analysis was initiated adhering to the exact time schedule of events utilized in the GC-HRMS MIS analyses. The reference mass and the exact mass range of interest were displayed alternately and were visible simultaneously on the MS oscilloscope. Precision and Accuracy of GC-HRMS Techniques. The precision and accuracy of capillary column GC-HRMS analyses were dependent on: (1)stability of MS electronic circuits; (2) sample preparation procedure (specificity and efficiency); (3) precise dilution or concentration of 60-pL sample extracts; (4) precise injection volumes; (5) capillary column resolution of components. The MS, when adjusted for ca. 7500 mass resolution and used as a GC detector in GC-HRMS MIS analysis, provided positive responses for those components which eluted from the GC and produced a molecular ion or fragment ion within f30 millimass units of TCDD masses m / z 319.8965, m / z 321.8936, and 37ClTCDD mass, m / z 327.8847. The intensity of the MIS response for a component other than TCDD was dependent on the difference in exact masses (i.e., only a fraction of the total intensity of a component mass differing from TCDD by f 3 0 millimass was detected in MIS analysis). Therefore, interference from contamination, chlorinated compounds, hydrocarbons, etc., was minimized or not detected. The MIS responses for 37C1-'EDD and TCDD were reproducible and linear relative to the amount injected. Two TCDD concentration ranges, 0.2 to 2 pg and 2 to 10 pg, were utilized to provide the most efficient and accurate quantification of TCDD residues. Sample extracts (60 pL) were concentrated or diluted as required for quantification purposes. The reproducibility of peak height responses during daily operation was observed to be *20% relative to 1 pg/pL or 5 pg/pL TCDD quantification standards. The TCDD m / z 320 and m / z 322 chlorine isotope ranged from 0.75:l to 0.98:l. The variation from the theoretical chlorine ratio, (0.76:1),was attributed to MIS integration rate, narrow bandwidth of capillary column peaks, and the small amount of TCDD analyzed in the presence of minor amounts of contamination. Because of the widespread distribution of PCBs, the accuracy of 37C1-TCDDdetermination primarily depends on the sample preparation efficiency and specificity, and the capillary column GC resolution of components. The MS mass resolution, ca. 45000, required to separate 37C1-TCDDm / z 327.8847 and PCB mlz 327.8775 is not feasible owing to MS limitations. For occasional and highly contaminated sample extracts, the 37C1-TCDDm / z 328 peak height was determined utilizing the PCB m / z 326 peak height to calculate the PCB contribution to the m / z 328, a mixture of 37C1-TCDDand PCB. The GC/HRMS peak matching accuracy for known elemental compositions was determined to be f 2 millimass units at 9500 mass resolution with PFK as reference. The PFK reference mass, m / z 318.9793, and TCDD mass were observed t o be exactly superimposed on the MS oscilloscope in real time at the exact GC-HRMS retention time of TCDD. The TCDD isomers were also confirmed in these analyses. The equation shown below was used to calculate the mean % accuracy for QA samples. % Accuracy = f [ l x , - x ^ l / x ^ ] (100) (per individual analysis)
ANALYTICAL CHEMISTRY, VOL. 52, NO 8, JULY 1980 n
[Ix,
Mean % Accuracy =
f
i=l
-
x^l/x^ ( l O O ) l ,
n (for all analysis performed)
Accuracy = the difference between an individual value x, (where x i = TCDD detected) and the true or correct value of the quantity measured 2 (where x^ = TCDD fortification level) Quality Assurance (QA) Program. The sample preparation laboratory assigned identification numbers to all samples. The samples and QA samples were fortified with 2.5 to 10 ng of 37C1-TCDDprior to analytical extraction and cleanup. The QA samples were also fortified with 0 to 1250 pg of TCDD. A method blank was included as part of the $A sample package. The sample extracts and quantification standards, 37C1-TCDDand TCDD, were submitted to the GC-MS laboratory in a blind fashion (Le. there was no way to distinguish between $A and actual samples). The efficiency, accuracy, precision, and validity of ppt TCDD analyses are dependent upon the incorporated quality assurance program. Analytical Extraction and Clean-up Procedures. Reagents. The hexane, acetone, benzene, methylene chloride, and acetonitrile were Mallinckrodt nanograde. The carbon tetrachloride was Fisher ACS grade (water, 0.01% maximum) and the ethyl alcohol was Matheson Coleman and Bell, pesticide quality. The alumina was Woelm neutral activity grade I. The Florisil, Floridin Co., was from a lot suitable for pesticide residue work. The sodium carbonate, sodium sulfate, potassium hydroxide, and sulfuric acid were Mallinckrodt AR grade. The sodium carbonate, and sodium sulfate were Soxhlet-extracted overnight with methylene chloride and dried at 200 " C . The water was passed through a column of activated carbon and distilled. The nitrogen used for solvent evaporation was Zero Grade obtained from Liquid Air, Inc., New Orleans, La. All solvents and chemicals were checked to verify the absence of background contamination. The labeled 2,3,7,8-tetrachlorodibenzo-p-dioxin having an isotopic purity greater than 98% 37C14was obtained from EcoControl, Inc. (71 Rogers Street, Cambridge, Mass. 02142). Analytical standards of 2,3,7,8-TCDD were obtained from Dow Chemical Co. (Midland, Mich.), IIT Research Institute (3441 So. Federal Street, Chicago, Ill. 60616), and Eco-Control, Inc. Preparation of Chromatographic Columns. Alumina chromatographic columns were prepared by packing 4.5 cm of neutral alumina into a clean and dry 53/4-inch (0.5-cm i.d.) disposable Pasteur pipet. The alumina was topped with 0.5 cm of anhydrous granular Na2S04. The column was washed with 4 mL of methylene chloride and residual solvent was forced from it using a stream of dry nitrogen. The columns were stored in an oven at 225 "C (minimum of 24 h) and were equilibrated to room temperature prior to use by placement in a desiccator over Drierite. Florisil chromatographic columns (11 X 500 mm) were packed with 15 g of 60-120 mesh Florisil (activated at 225 "C for 24 h). The column was topped with 2.5 cm of anhydrous granular sodium sulfate and pre-washed with 100 mL of hexane. Sample Preparation Using Acid/Base Procedure. Fish and L e a n Tissue. Tissue samples were ground to obtain a homogeneous sample. A 10 to 20 g sample was weighed into a 100-mL boiling flask. Five to 10 ng of 37C1-TCDD standard solution, 20 mL of ethyl alcohol, and 40 mL of 45% potassium hydroxide solution were added. The flask was heated under reflux with stirring for 2.5 h. After cooling, the solution was quantitatively transferred to a separatory funnel and the flask was rinsed with 10 mL of ethyl alcohol followed by 20 mL of hexane. The solution was extracted with four 25-mL portions of hexane and the hexane extracts were combined. Adipose Tissue. The samples were ground or rendered if necessary to obtain a representative sample that was free from connective and other tissue. Ten to 20 g of tissue were fortified with 5 to 10 ng 37C1-TCDDand 15 mL of distilled water was added. The mixture was refluxed with aqueous KOH/ethanol and extracted as described in the lean tissue procedure. Milk. Two and one-half ng of 37C1-TCDDstandard solution were added to 10 to 20 g of milk. The sample was refluxed with aqueous KOH/ethanol and extracted as described in the lean tissue procedure.
1241
Water. One kg of a well shaken water sample (including particulate matter, if present) was fort,ified with 2.5 ng 37C1-TCDD standard solution and extracted with three 100-mL portions of methylene chloride. The combined methylene chloride extracts were evaporated to dryness through a Snyder column utilizing a steam bath. One hundred mL of hexane were added to the residue and the resultant hexane was washed with 50 mL of 1 N KOH solution followed by concentrated sulfuric acid as described in the clean-up section. Soil and Sediment. Ten to 20 g o f a well mixed sample was fortified with 2.5 ng 37C1-TCDD,refluxed with aqueous KOH/ ethanol, and extracted as described in the lean tissue procedure. After refluxing and cooling, the solution was decanted into a separatory funnel through a filter funnel packed with glass wool. The boiling flask and the fiiter funnel were rinsed with two 10-mL portions of ethyl alcohol followed by 20 mL of hexane. The solution was then extracted with four 25-mL portions of hexane which had been previously used to rinse the boiling flask and filter funnel. The combined hexane extracts were then subjected to the acid/base clean-up procedure. Clean-up Procedure f o r AcidlBase Extract:;. The combined hexane extracts obtained as described in the sample preparation and extraction section were washed with 25 mL of 1 N KOH solution followed by four 50-mL portions of Concentrated sulfuric acid. After a 25-mL water wash, the water and hexane layers were neutralized by the addition of powdered Na2C03. The aqueous layer was discarded, and the hexane was dried by passage through a 11 mm i.d. X 500 mm glass column containing 10 cm of anhydrous powdered Na2C03. The hexane was collected in a Kuderna-Danish evaporative concentrator and concentrated to a volume of 3 mL. This concentrate was transferred to an alumina column that had been pre-wetted with 1 mL of hexane. The column was eluted with 6 mL of carbon tetrachloride (discarded) followed by 4 mL of methylene chloride (collected in a 12-mL distillation receiver). The receiver was capped with a micro-Snyder column. A carborundum boiling chip was added and the methylene chloride was evaporated just to dryness by means of a hot water bath. Two separate 2-mL portions of hexane were added to the distillation receiver and each was evaporated just to dryness. The residue was dissolved in 3 mL of hexane and was chromatographed on a second alumina column as just described. The methylene chloride eluant from the second alumina column was evaporated just to dryness. Two mL of benzene were added to the receiver and the solution was concentrated to a volume of 100 pL. The benzene solution was transferred quantitatively to a 2-mL graduated chromaflex sample tube (Kontes Glass Company K-422560). Using a slow stream of dry nitrogen, the benzene solution was carefully concentrated to a volume of 60 wL. This extract was sealed in glass tubing (3 mm i.d. X 7 em) and was stored at sub 0 "C until analysis by mass spectrometry. Sample Preparation Using Neutral Procedure. Fish Tissue. Tissue samples were ground to obtain a homogeneous sample. A 15-g sample and 150 g of anhydrous granular sodium sulfate were placed into a Waring blender jar and the mixture was blended for 1 min. The mixture was then blended with 50-g portions of dry ice until the sample was thoroughly powdered. The powder was transferred to a flask and 10 ng of 37C1-TCDD were added directly onto the powder. The blender jar was rinsed with acetonitrile, and the rinsings plus enough additional acetonitrile to make up exactly 150 mL were added to the flask. After vigorous magnetic stirring for 2 h the mixture was filtered through a glass filter tube (42 X 160 mm) containing approximately 30 g of anhydrous granular sodium sulfate. Clean-Up Procedure for Neutral Extract. Exactly 100 mL of the acetonitrile extract (representing 10 g of sample) obtained in the previous section were partitioned (12) with 50 mL of acetonitrile-saturated hexane. The hexane was then partitioned with two 100-mL portions followed by one 50-mL portion of hexane-saturated acetonitrile. All acetonitrile layers (350 mL total) were combined and re-partitioned with 10 mL of acetonitrilesaturated hexane. Using a Snyder column, the acetonitrile was concentrated to a volume of 10 mL on an explosion-proof hotplate. The residue was taken up in two separate 100-mL portions of hexane and each was concentrated to a volume of 5 to 10 mL. The hexane residue was transferred to a Florisil column, using three 5-mL portions of hexane. The column was washed with
1242
ANALYTICAL CHEMISTRY, VOL. 52, NO. 8 , JULY 1980
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Table I. Criteria Utilized for Confirmation of 2,3,7,8-TCDD Residues
,
01
1.
2. 3.
4.
5.
Correct capillary column GC-HRMS retention time of 2,3,7,8-TCDD. Correct chlorine isotope ratio of the molecular ion ( m / z 320 and m/z 322). Correct capillary column GC-HRMS multiple ion monitoring response for TCDD masses and 37ClTCDD mass (simultaneous response for elemental composition of m/z 320, m/z 322, and m/z 328). Correct response for co-injection of sample fortified with 37C1-TCDDand TCDD standard. Response of m/z 320 and mlz 322 must be greater than 2 . 5 times millimeters of noise level.
Supplemental criteria which may be applied t o samples from highly contaminated sources (A) (B)
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100 mL of 10% methylene chloride in hexane (v/v) and the washings were discarded. The column was eluted with 100 mL of 25% methylene chloride in hexane (v/v). The eluate was collected in a Kuderna-Danish evaporator and was concentrated to a volume of 3 mL utilizing a steam bath. The residue was dissolved in 100 mL of hexane and was concentrated to a volume of 3 mL. The hexane residue was transferred to an alumina column and cleaned up as described in the acid/base procedure with the exception that only one alumina column was used. DISCUSSION AND RESULTS Glass capillary columns enhanced the GC-HRMS MIS method of analysis by (1) providing the required resolution of complex mixtures into individual components before they entered the MS; (2) the narrow band width of the TCDD component enhanced M S sensitivity; (3) direct coupling of capillary column to MS ensured maximum transfer efficiency; (4) capillary column bleed rate was low; therefore, background contamination was minimized and the MS sensitivity was enhanced. The requirements imposed on the MS utilized as a GC detector in these analyses were (1) extremely stable electronic circuits; (2) ultra high sensitivity; (3) specific mass detection. The requirements were satisfied by optimizing all components that influenced sensitivity, noise, and mass resolution. T h e MIS response for a quantification standard, 37C1-TCDDa n d TCDD is shown in Figure 1. Samples collected from highly contaminated sources, i.e., chemical dump sites or fish from contaminated rivers, present serious problems. T h e high concentrations of chlorinated compounds overload the acid/ base clean-up procedure and many compounds are co-extractable with TCDD. These components cause capillary column overload, co-elution of components, etc. T h e highly specific neutral clean-up procedure was developed and applied to a number of these samples t o provide additional confirmation of TCDD residues. It was very effective for this specific isolation of TCDD from other chlorinated components but has not been validated for quantitative TCDD analysis using multiple laboratory participation. Q u a n t i f i c a t i o n of T C D D . T h e MIS simultaneous peak
COCl loss indicative of TCDD structure Capillary column GC-HRMS peak-matching analysis of mlz 320 and mlz 322 in real time t o confirm the TCDD elemental compositions.
height responses, m / z 328, m l t 322, and m / z 320, of sample and the sample fortified with known amounts of 37C1-TCDD and TCDD were utilized to determine the percent recovery of 37C1-TCDD(sample preparation efficiency), TCDD residue level, and limit of detection. T h e experimental 37C1-TCDD percent recovery value was used to correct the TCDD residue level and limit of detection for losses in the sample preparation efficiency. A minimum acceptable percent recovery value (50%) for 37C1-TCDDwas established for reporting TCDD analysis. TCDD results were not corrected for recovery values greater than 100%. The occasional 37C1-TCDDrecovery value between 100% and 135%, was attributed to interference from PCBs and unidentified contamination. Typical concentrations of quantification standards were 50 to 300 pg/kL 37C1-TCDD and 1 to 5 pg/pL TCDD. Limit of Detection. The minimum limit of detection was defined as the amount of TCDD that would provide clearly defined peak shapes ( m / z 320 and m / z 322) in the proper isotopic ratio and with a signal-to-noiseratio greater than 2.5:l. T h e sample weight, size, percent recovery, sample matrix effects, and electronic noise present in the time frame of measurement does affect the detection limit. C r i t e r i a f o r C o n f i r m a t i o n of T C D D Residues. T h e capillary column GC-HRMS MIS analysis for 2,3,7,8-TCDD residues must satisfy the criteria, 1-5, shown in Table I to be considered a confirmed positive sample. The supplemental criteria, A and B, were occasionally applied t o samples collected from highly contaminated sources. P u r i t y of 37C1-TCDDFortification S t a n d a r d . A TCDD isomer satisfying the analytical criteria for 2,3,7,8-TCDD (correct retention time and elemental composition) was detected a t a concentration level of l pg/ng 37C1-TCDD. These results indicated erroneous sub-ppt TCDD results would be generated for 10-g samples fortified with 10 ng 37C1-TCDD and this was confirmed experimentally utilizing human milk. The 37C1-TCDDfortification level was reduced to 2.5 ng t o avoid false positive results and provide more accurate quantification of sub-ppt TCDD analyses. H u m a n Milk Studies. The samples were subjected to the described acid/base extraction and clean-up procedure prior to GC-HRMS MIS analyses. The 60-pL human milk extracts were quantitatively concentrated to 7-20 pL utilizing dry nitrogen gas for sub-ppt TCDD analysis. T h e MIS analysis sequence was sample, and the sample fortified with 37ClTCDD and TCDD. A typical analysis for a QA sample of human mother's milk is shown in Figure 2. The experimental results indicated the sample contained 1.2 ppt TCDD residue. This 10-g sample had been fortified with 10 pg (1 ppt) of
ANALYTICAL CHEMISTRY, VOL. 52, NO. 8 , JULY 1980 I
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Flgure 2. Capillary column GC/HRMS multiple ion selection chromatogram obtained from an extract of QA human milk: (A) 2 I.LLof sample; (B) 1 pL of sample fortified with 37CI-TCDDand TCDD quantification standard (QA fortification level, 1 ppt TCDD; experimental result, 1.2 ppt TCDD)
Figure 3. Capillary column GC/HRMS multiple ion selection chromatogram obtained from an extract of ocean perch: (A) sample; (B) sample fortified with 37C!-TCDD and TCDD quantification standard. The sample had been fortified with 5 ng 37CI-TCDDand 37 ppt TCDD. The experimental results were: 50% recovery of 37CI-TCDD;3 4 ppt TCDD detected: 4 ppt detection limit
Table 111. Typical Analytical Results for Quality Assurance Samples Generated during t h e Analysis of Fish for 2,3,7,8-TCDD Residues
Table 11. Typical Analytical Results for 2,3,7,8-TCDD Residues in Quality Assurance Samplesa of Human Milk experimental results 37 c1TCDD TCDD detection TCDD % limit.b detected.b recovery PPt PPt 50 0.3 1.9 0.6 72 0.2 68 0.1 0.2 64 0.3 ND 0.9 68 0.4 1.4 84 0.2 64 0.2 0.4 51 0.2 ND 0.6 73 0.4 1.4 52 0.3 3.0 72 0.5 0.5 4.0 100 5OC 0.2 ND
TCDD fortification level Pg 10 3 1 0 9 20 5 2 7.5 6.5 30 50 0
PPt 1.0 0.3 0.1 0 0.9 2.0
0.5 0.2 0.75 0.65 3.0 5.0 0
Each 10-gm sample was fortified with 2.5 ng "ClTCDD. Corrected for % recovery losses. Method blank. ND = not detected, 37C1-TCDDmean % recovery, 68%. TCDD mean % accuracy, k 3 8 % . a
TCDD. T h e total TCDD analyses (analytical clean-up efficiency, TCDD residue level, and limit of detection) were performed on two sample injections: sample, and fortified sample. Duplicate analyses were usually performed on each sample. T h e results of a quality assurance study incorporating human milk fortified with 2.5 ng 37C1-TCDD and 0 to 5 ppt TCDD are shown in Table 11. Evaluation of the experimental results and TCDD fortification level indicate: (1)The sample preparation procedure and MIS method of analysis provided reasonably accurate TCDD analyses in the 0.2 to 5 ppt concentration range considering the amount of TCDD present; (2) False positive results were not detected; (3) 2.5 ng 37ClTCDD fortification level was adequate for determining the sample preparation efficiency; (4) T h e 37C1-TCDDcontamination (2.5 pg TCDD) and corrections for sample preparation efficiency exhibit significant effects in 0 to 1 p p t analyses. Based on recent experiments, the accuracy of 1 p p t TCDD
experimental results sample weight, g, and ID
TCDD detection TCDD % limit, detected, recoverya pptb pptb 62 2 20 52 4 34 82 3 ND 100 3 ND 54 1 19 100 2 ND 78 2 ND 92 2 19 97 5 45 37ClTCDD
TCDD fortification level
Pi? PPt 110 22 5 185 35 5 (1) 0 0 5 (1) 0 0 5 (4) 70 14 5 (3) 0 0 5 (3) 0 0 5 (3) 55 11 5 (1) 240 48 5 (1) loo+ 1 8 130 13 1 0 (1) loo+ 4 43 600 60 10 (1) loo+ 7 ND 0 0 10 ( 2 ) loo+ 3 ND 0 0 1 0 (2) loo+ 4 76 1250 125 10 (1) 1 ND 0 0 67 10 (4) 93 3 56 6 50 65 10 (1) 10 (1) 84 4 73 620 62 a Each sample has been fortified with 5 or 10 ng V l TCDD. Corrected for % recovery losses, ND = not detected. 37C1-TCDDmean % recovery, 86%, TCDD mean %accuracy, +15%. (1)Ocean perch, (2) lake trout, ( 3 ) beef liver, ( 4 ) method blank. analyses can be improved when the 37C1-TCDDfortification level is reduced to 0.5 to 1 ng. Fish, Water, a n d Sediment Analyses. The samples were subjected to the described preparation procedure. A GCHRMS MIS analysis of a fish extract and the extract fortified with 37C1-TCDDand TCDD quantification standard is shown in Figure 3. Unusual and high concentrations of chlorinated contaminate masses differing from the exact mass of TCDD were detected in fish collected from polluted waters. The high concentration of co-extractable chlorinated components in fish caused serious problems. T o minimize and/or cancel the effects, a small sample size and very high MS sensitivity was utilized. T h e analytical results generated for QA samples during these studies are shown in Table 111. Evaluation of QA results indicate reasonably accurate TCDD results were
1244
ANALYTICAL CHEMISTRY, VOL. 52, NO. 8, JULY 1980
Table IV. Typical Analytical Results for Quality Assurance Samples Generated during the Analysis of Water and Sediment for 2,3,7,8-TCDD Results experimental results TCDD TCDD -C1detecfortification sample TCDD,a tion TCDD level limitb detected.b weight, g, % a n d 1 ~ - recovery ppt ppt ' Pg PPt 1000 ( 1 ) C 73 0.02 0.09 75 0.08 1000 (1) 90 0.05 0.7 1000 1.0 1000 (1) 66 0.01 ND 10 0.01 1000 (1) 95 0.01 0.05 50 0.05 1000 (1) 78 0.02 0.1 100 0.1 1000 (1) 78 0.02 0.03 25 0.03 lOOO(1) 100+ 0.04 5-00 0.5 0.4 69 0.13 1.0 50 1.0 50 ( 2 ) 0.14 1.0 100+ 70 1.4 5 0 (2) lO(2) 100+ 0.6 2.5 16 1.6 10 ( 2 ) 96 0.5 3.3 46 4.6 lO(2) loo+ 2.0 23.0 170 17.0 10 ( 2 ) 68 4.0 30.0 350 35.0 lO(2) 100 0.7 ND 0 0 a Each sample had been fortified with 2.5 ng 37ClTCDD. Corrected for % recovery losses. ND = not detected. "Cl-TCDD mean % recovery, 87%. TCDD mean % accuracy, i: 16%. (1)water, ( 2 ) sediment, obtained and no false positive results were detected. T h e number of QA samples with zero fortification levels of TCDD were very important because of the unusual and high concentrations of chlorinated contamination. The supplemental criteria, Table I, provide additional confirmation of TCDD residues in the presence of high concentrations of chlorinated contamination. T h e analytical results for QA samples, Table IV, were generated during water and sediment studies. Reasonably accurate results were obtained for 0.03 ppt to 1 ppt TCDD in water and 0.5 ppt t o 35 ppt TCDD in sediment. Water extracts were very clean. Significant amounts of contamination differing from the exact mass of TCDD were detected in specific sediment extracts but did not interfere with TCDD analysis. T C D D Isomers. Recent reports (13-16) have shown that chlorinated dioxins may be formed in combustion processes. T h e toxicological properties of the known isomers of TCDD are significantly different; however, the mass spectra are almost identical except in the low mass range (9),and the minor difference is not significant in ppt analysis of environmental and biological extracts. Therefore, it is extremely important that TCDD isomers be resolved utilizing highly efficient glass capillary columns before introduction into the MS in order t o provide more conclusive identification. The 2,3,7,8-TCDD and the available TCDD isomers may be separated, detected, and/or quantified utilizing the described technique. Unfortunately only a limited number of the 22 TCDD isomers are commercially available. The TCDD isomers utilized in this laboratory for co-injection purposes are 1,2,3,4-, 1,3,6,8-, 2,3,6,8-, and 2,3,7,8-TCDD. The described SE-30 WCOT glass capillary column resolution of TCDD isomers and order of elution was similar to the work reported by Buser in which he used OV-101 glass capillary column GC-MS for chromatographic separation of seven out of nine TCDD isomers. This work showed t h a t two out of eight other TCDD isomers coelute with 2,3,7,8TCDD on OV-101 and suggests that no single capillary column will be able to separate all 22 TCDD isomers. Confirmation of 2,3,7,8-TCDD in the presence of other TCDD isomers also requires analysis on a second capillary column of different polarity t o differentiate between isomers. Preliminary studies utilizing the acid/base clean-up pro-
cedure and nanogram quantities of hexa, hepta, and octa substituted dibenzo-p-dioxins (analytical standards) suggest tetra chlorodioxin isomers are not formed from the degradation of higher chlorinated dioxins by the acid/base sample preparation conditions. Also nanogram quantities of 2,4,5trichlorophenol showed no evidence of condensation to the 2,3,7,8-TCDD under the same acid/base sample preparation conditions. C o n t a m i n a t i o n . Utilizing the criteria shown in Table I for confirmation of TCDD residues, chlorinated compounds do not present significant problems in TCDD analysis except in 0 to 5 ppt analysis or in rare cases of gross contamination. Neutral clean-up procedures, mass resolution of 14 000, and polar or nonpolar capillary columns effectively resolve this type of occasional problem. In general, PCBs were the contamination of major concern. The mass resolutions ca. 12000 and 45000 required to separate PCB masses 321.8677, 327.8775 from TCDD masses 321.8935, 327.8847 could not be utilized in 0 to 5 ppt analysis due t o limitations in instrument design a n d sensitivity. T h e P C B interference had obvious effects: (1)Recovery of 37C1-TCDD was greater than 100%; (2) T h e TCDD m / z 320, m / z 322 chlorine isotope ratio was destroyed. Corrections for P C B interference could be applied if necessary but a t a sacrifice in minimum detection limit. T h e mass resolution, 5000 t o 9000 was sufficient for most TCDD analyses. The occasional and unexpected sample containing high ppt to ppm levels of TCDD may cause serious contamination problems (glassware, etc.) to other TCDD samples prepared in the laboratory. Erroneous and low ppt TCDD residues may then be detected in succeeding samples as a result even though extremely meticulous glassware cleaning procedures are exercised. Laboratory records, good quality assurance practices, and multiple laboratory participation should detect and/or eliminate these problems. ACKNOWLEDGMENT We thank the following people for their help in various stages of this project: James Gibson and Henry Shoemaker, EPA-Bay St. Louis, lab, for extraction and clean-up of samples, and for suggestions resulting in improvement of extraction and clean-up methodology. Mike Dellarco, Thomas Holloway, Carolyn Offutt, and Richard Reising, EPA-Washington, D.C., for their help in the overall coordination of the dioxin program. Use of trade names is for identification only and does not constitute endorsement by the U.S. Environmental Protection Agency. LITERATURE CITED (1) "Report on 2,4,5-T", A report of the Panel on Herbicides of the President's Science Advisory Committee, Executive Office of the President, Office of Science and Technology, March 1971. (2) Young, A. L.; Calcagni, J. A.; Thalken, C. E.; Tremblay, J. W. "The Toxicology, Environmental Fate, and Human Risk of Herbicide Orange Air Force and Its Associated Dioxin", Technical Report OEHL TR-78-92, Occupational and Environmental Health Laboratory, Brooks Air Force Base, Texas, October 1978. Perspective on Chlorinated Dibenzodioxins and Dibenzofurans, Experimental Issue No. 5,National Institute of Environmental Health Sciences, Research Triangle Park, N.C., September
1973. (3) Sparschu, G. L.; Dunn, F. L.; Rowe, V. K. FoodCosrnet. Toxicol. 1971, 9 . 405. (4) Baughman, R. W.; Meselson, M. S. Environ. Health Perspectives 1973, 5 , 27. (5) O'Keefe, P. W.; Meselson, M. S.; Bauahman, R. W. J . Assoc. Off. Anal. Chern. 1978, 61, 621. (6) Shadoff, L. A.; Lamparski, L.; Davidson, J. H. Bull Environ. Contarn. Toxicol. 1077. 18. 478. (7) Shadoff, L. A.f Hummel, R. A. Biorned. Mass Spectrorn. 1978. 5 , 1. (8) Harless, R. L.; Oswald, E. 0. "Dioxin, Toxicological and Chemical Aspects". Cattabini, Flaminio, Cavallaro, Aldo, Galli, Giovanni, Eds.; Spectrum Publications, Inc.; 1978;pp 51-57. (9) Buser, H. R. Anal. Chem. 1977, 49, 918. (IO) Hass, J. R.; Freiesen, M. D.: Harvan, D. J.; Parker, C. E. Anal. Chern. 1978, 50, 1474. (1 1) Harless, R. L.; Ellis, P. R.; Oswald, E. O., 26th Annual Conf. on MS and Allied Topics, 1978; paper no. RP-7.
Anal. Chem. 1980, 52, 1245-1248 (12) Mills, P. A. J. Assoc. Off. Anal. Chem. 1961, 4 4 , 171. (13) Chlorinated Dioxin Task Force, Michigan Division Dow Chemical U.S.A., "The Trace Chemistries of Fire-A Source for the Entry of Chlorinated Dioxins into the Environment". 1978. (14) Buser, H. R.; Bosshardt, H. P.; Rappe, C. Chemosphere 1978, 165. (15) Olie, K.; Vermeulen, P. L.; Hutzinger, 0. Chemosphere 1977, 455. (16) Rappe, C.; Buser, H. R.; Bosshardt, H. P. The 23rd Collaborative In-
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ternational Pesticide Analytical Council (CIPAC) Symposium, Baltimore, Md., June 1979.
RECEIVED for review September 4, 1979. Accepted February 28, 1980.
Pyrolysis Gas Chromatographic-Mass Spectrometric Identification of Polydimethylsiloxanes John C. Kleinert' and Charles J. Weschler" Bell Laboratories, Holmdel, New Jersey 07733
Pyrolysis of polydimethylslloxanes (980 O C , 1 s) yields a series of cyclic dimethylslloxanes that are separated and detected using gas chromatography-mass spectrometry. Trace amounts of polymer can be quantitatively analyzed by selectively monitoring the Isotopic cluster of ions at m / e 207, 208, and 209 present in the mass spectrum of the major pyrolysis product, hexamethylcyclotrisiloxane. Application of this technique to polydlmethylsiloxane determination in the range from to lo-'' g Is demonstrated. The amount of detected materials increases as the viscosity of the fluid increases, and this limits accuracy to about an order of magnitude. However, if the viscosity of the polydlmethylsiloxane is known, the technique permits analyses accurate to within 10%.
Approximately 235 million pounds of silicones were produced in 1978 ( I ) , almost one tenth the amount of nylon produced during the same year (2). Silicones are found in a vast array of products, including synthetic lubricants, paint resins, polishes, coolants, fuser oils, plastics, brake fluids, sealants, surfactants, and dielectrics. Polydimethylsiloxanes are easily the largest class of silicones produced. These materials have low surface tensions, about 17-22 dynes/cm, and spread or creep over all types of surfaces ( 3 ) . As a consequence, they are particularly troublesome contaminants that can destroy adhesion, make repainting or refinishing difficult, interfere with the wetting of solders, and even stress crack polyethylene. Creepage is of special concern to the electronics industry, where silicone contamination on operating electrical contacts can lead to equipment failures (4).An analytic capability to detect such contamination is obviously valuable. A further application of trace silicone analyses is suggested in a recent report by Pellenberg (5). Silicones are totally synthetic, ubiquitous, and possess excellent thermal and chemical stability. Given the above, silicones may serve as sensitive tracers for anthropogenic additions to the environment. This report describes the application of pyrolysis gas chromatography-mass spectrometry to the detection of polydimethylsiloxanes. Fluids ranging in viscosity from 20 to 30 000 centistokes have been examined. Pyrolysis products have been identified, and correlations have been made between Summer research student. Present address: Department of Chemical Engineering, Princeton University, Princeton, N.J. 08544. 0003-2700/80/0352-1245$01 .OO/O
the amounts of major products and the amount of starting material. As little as 0.1 ng of a given polydimethylsiloxane has been detected using the described techniques. EXPERIMENTAL Equipment and Procedures. The pyrolysis studies were performed using a Chemical Data Systems "Pyroprobe 100" interfaced to a Hewlett-Packard 5992A gas chromatograph-mass spectrometer equipped with a single stage jet separator. The pyrolysis products were separated on a 1.22 m X 2.0 mm i.d. glass column packed with 1%SP-2250 on 10G120 mesh Chromosorb W-HP. The GC oven temperature was held at 50 "C for 1 min and then programmed to 220 "C at 8 "C per min. The injection port was held at 150 "C, the pyrolysis interface was held at 100 "C, and the helium carrier gas flow was maintained at 20 cm3/min. A platinum ribbon probe was used for the majority of the pyrolysis studies, but a few experiments were performed using a coil probe and a quartz sample tube. Pyrolyses were carried out with rise times of approximately 75 "C/ms for the ribbon element and 1 "C/ms for the coil element, an upper temperature limit of 980 "C (unless otherwise stated), and total pyrolysis times of 1 s for the ribbon probe and 5 s for the coil probe. Reported temperatures are corrected final temperatures. In a typical run, 5.0 pL of silicone-containingsolution were spread evenly over the platinum ribbon. The ribbon was fired twice at 70 "C (10 s) to remove solvent and then sealed in the pyrolysis interface for a minimum of 15 min (for air to be purged) before initiating the run. Materials. Linear polydimethylsiloxanes ranging in viscosity from 20 to 30000 centistokes were obtained from a number of suppliers including Dow Corning, General Electric. Union Carbide, Harwick, and Petrarch Systems. Silicone solutions of known concentration were prepared using freshly distilled 1,2,2-trifluoro-1,2,2-trichloroethane(Freon 113). When using the coil probe, the samples were placed in quartz tubes 2 mm X 25 mm. These tubes were rinsed with distilled Freon 113 and air fired at 900 "C to ensure cleanliness before use. A similar procedure was used to clean the platinum ribbon (35 mm X 1.5 mm X 0.0127 mm) prior to each analysis with the ribbon probe. RESULTS A N D D I S C U S S I O N A series of exploratory pyrolyses were conducted with 30 000 centistoke polydimethylsiloxane to determine optimum operating conditions. With a fixed total pyrolysis time of 1 s, 5.0-pL samples of 0.04% polydimethylsiloxane solutions were pyrolyzed a t upper temperature limits of 700, 800,900, and 980 "C. The higher the final temperature, the more abundant the pyrolysis products detected, although the relative distribution of pyrolysis products remained essentially unchanged. With a fixed upper temperature of 980 "C, the total pyrolysis time was varied from 0.1 to 5 s. The abundance of detected products increased with increasing time intervals for t 2 1980 American
Chemical Society
